Transcript Analysis of the Basal Section of Leaves Suggests Delayed Plastid Development and Perturbation of Plastid Gene Expression in WHY1 Knockdown Lines
In wild type leaves, transcripts associated with key developmental processes were highly abundant in the basal section of leaves and declined in the middle and tip sections. We therefore conducted one-way ANOVA to determine significantly differentially abundant transcripts in the base of wild type, W1-1 and W1-7 leaves. This identified 1267 transcripts that were differentially abundant dependent on genotype of which 540 were not assigned a function (Table S5). The remaining 727 transcripts were assigned to a range of functions associated with primary and secondary metabolism, RNA, DNA and protein processing, cellular organisation and development (Table S5).
Seventy-six transcripts encoding proteins associated with plastid biogenesis and development were differentially expressed, (Table S6; Fig 6) the majority of which were more abundant in the base of 7-day old leaves of WHY1 knockdown plants than the wild type. These included transcripts encoding elements of the photosynthetic electron transport chains and Calvin cycle proteins including Rubisco (Table S6). Furthermore, several transcripts (AK248405.1, MLOC_39198.3, MLOC_52167.2 and MLOC_63408.1) homologous to Arabidopsis transcripts encoding proteins required for FeS cluster assembly (Bastow, Bych, Crack, Le Brun, & Balk, 2016; Hu, Kato, Sumida, Tanaka, & Tanaka, 2017; Roland et al., 2020; Xu, Adams, Chua, & Møller, 2005;) were less abundant in the wild type than WHY1. Many transcripts encoded proteins associated with the targeting and translocation of proteins to the chloroplast as well as several proteins associated with protein folding such as AK250352.1 and AK361117 which both exhibit homology to AT3G60370 encoding an immunophillin with protein folding activity required for the assembly of PSII (Lima et al., 2006) and AK251420.1 encoding a chaperonin homologous to AT2G28000 required for plastid division (Suzuki et al., 2009). Transcripts homologous to Arabidopsis transcripts encoding several plastid localised Clp proteases were also present including several (MLOC_32972.1, MLOC_4257.1, MLOC_64141.1, MLOC_68297.2, MLOC_861.2) that were previously demonstrated to play a role in plastid biogenesis (Kim et al., 2009).
A small number of transcripts exhibited a pattern of low abundance in W1-7. These included two transcripts (MLOC_56051.1, MLOC_56052.1) similar to AT2G34420 encoding a chlorophyll a-b binding protein, MLOC_61567.1 similar to plastid encoded ATCG01010 encoding an NADH dehydrogenase subunit. Interestingly, the levels of two transcripts (MLOC_65876.1, AK353571) with similarity to an Arabidopsis transcript (AT2G20570; GLK1) encoding a transcription factor required for the expression of nuclear encoded photosynthetic genes (Waters et al., 2009) were lower in all sections of the WHY1-7 leaves.